Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity.
Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity.
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DOI：
10.1002/cm.20480
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发表时间：
2010-11
期刊：
影响因子：
2.9
通讯作者：
Cowan, Nicholas J.
中科院分区：
文献类型：
--
作者：
Tian, Guoling;Thomas, Simi;Cowan, Nicholas J.
α/β微管蛋白异二聚体的组装需要一系列伴侣蛋白（TBCA-E）的参与，这些伴侣蛋白在胞质伴侣蛋白CCT下游发挥作用，作为异二聚体组装机器。TBCD和TBCE也能够在破坏天然异源二聚体的逆反应中起作用。TBCA-E的同源物存在于所有真核生物中，并且α-和β-微管蛋白同种型的氨基酸序列在脊椎动物中严格保守。然而，TBCD在体内影响微管蛋白破坏的效率取决于其来源：牛（而不是人）TBCD在培养细胞中过表达时有效地破坏微管蛋白和微管。在这里，我们表明，重组牛TBCD在HeLa细胞中作为与β-微管蛋白的化学计量的共复合物产生，与其在体外和体内的行为一致。相反，使用相同的宿主/载体系统表达人TBCD导致产生不与β-微管蛋白复合的TBCD。我们发现，重组人TBCD的功能与其非重组牛对应物在体外CCT驱动的折叠反应中，在微管蛋白破坏反应中，以及在微管蛋白GAP测定中，其中TBCD和TBCC在远低于聚合成微管所需的异二聚体浓度下刺激β-微管蛋白的GTP水解。我们得出结论，牛和人类TBCD具有功能相同的作用，从头微管蛋白异二聚体组装，并表明，人类TBCD无法破坏微管的完整性后，在体内过表达可以克服siRNA介导的抑制表达的TBCD调节Arl 2（ADP核糖基化因子样蛋白2）。
Assembly of the α/β tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin, CCT, as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and β-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric co-complex with β-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with β-tubulin. We show that recombinant human TBCD functions indistinguishably from its non-recombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GAP assays in which TBCD and TBCC stimulate GTP hydrolysis by β-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP Ribosylation Factor-like Protein 2).
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DOI：
10.1083/jcb.149.5.1087
发表时间：
2000-05-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Bhamidipati A;Lewis SA;Cowan NJ
通讯作者：
Cowan NJ
影响因子：
64.5
作者：
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通讯作者：
Cowan, NJ
DOI：
10.1073/pnas.91.19.9116
发表时间：
1994-09-13
影响因子：
11.1
作者：
VINH, DBN;DRUBIN, DG
通讯作者：
DRUBIN, DG
影响因子：
3.7
作者：
Lopez Fanarraga, Monica;Bellido, Javier;Carlos Zabala, Juan
通讯作者：
Carlos Zabala, Juan
DOI：
10.1083/jcb.138.4.821
发表时间：
1997-08-25
期刊：
The Journal of cell biology
影响因子：
--
作者：
Tian G;Lewis SA;Feierbach B;Stearns T;Rommelaere H;Ampe C;Cowan NJ
通讯作者：
Cowan NJ